Previous studies from this laboratory have led to the development of affinity chromatographic procedures for the purification of relatively large amounts of homogeneous, cosubstrate-free, crystalline dihydrofolate reductase from chicken liver. Thus sufficient enzyme protein from liver, the main site of dihydrofolate reductase activity in animals, was now available for sequencing. The S-carboxymethylated protein was subjected to cleavage by cyanogen bromide which produced five fragments. Sequences and ordering of the cyanogen bromide fragments were established by means of automated sequencer analysis of the fragments and from smaller peptides generated by proteolysis with trypsin and staphyloccal protease. The complete amino acid sequence comprising the covalent structure of the single polypeptide chain consists of 189 residues of molecular weight 21651.